Project Summary/Abstract Regulated proteolysis by the ubiquitin-proteasome system (ubiquitin system) plays essential roles in a multitude of biological processes and has major ramifications for human health and disease, including illnesses that range from cancer and neurodegeneration to cardiovascular syndromes and defects of immunity. Our studies of the ubiquitin-proteasome system and ubiquitin-dependent N-degron pathways (previously called ?N-end rule pathways?) over more than three decades were made possible, to a large extent, by the present grant (GM031530), currently in its 37th year of support. N-degron pathways recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, polyubiquitylate these proteins and thereby cause their degradation by the proteasome or autophagy. Recognition components of N-degron pathways, called N-recognins, are E3 ubiquitin ligases that can target N-degrons. One eukaryotic N-degron pathway, called the Arg/N-degron pathway, targets specific unacetylated Nt-residues of protein substrates. This pathway, discovered by our laboratory in 1986, continues to be a fount of biological insights. The Ac/N-degron pathway, discovered by our laboratory in 2010, recognizes proteins that bear N-terminally acetylated residues. The other two proteolytic systems, the Pro/N-degron pathway and the fMet/N-degron pathway, are mediated, respectively, by the E3s GID and Psh1, and recognize, respectively, the Nt-proline (Pro) and the Nt-formyl-Met (fMet) residues of protein substrates. This resubmitted GM031530 renewal application stems from our unpublished studies over the last ~2 years, contains results obtained the last ~10 months as well, and focuses on the mammalian Arg/N-degron pathway, including the recently discovered ability of its E3s Ubr1/Ubr2 to bind to transcriptional factors (TFs) Atf3, Fosl1, c-Fos, Fosl1, Prep1, and Prep2. Beginning with Atf3, we recently showed that, in vivo, the binding of Atf3 to Ubr1/Ubr2 results in the degradation of Atf3 by the Arg/N-degron pathway. These and other (related) studies, described in Specific Aims of the resubmitted GM031530 renewal application, will advance the understanding of protein degradation and the universally present (as well as medically significant) Arg/N-degron pathway.